Aberrant splicing of CaV1.2 calcium channel induced by decreased Rbfox1 enhances arterial constriction during diabetic hyperglycemia

Diabetic hyperglycemia induces dysfunctions of arterial smooth muscle, leading to diabetic vascular complications. The CaV1.2 calcium channel is one primary pathway for Ca2+ influx, which initiates vasoconstriction. However, the long-term regulation mechanism(s) for vascular CaV1.2 functions under hyperglycemic condition remains unknown. Here, Sprague–Dawley rats fed with high-fat diet in combination with low dose streptozotocin and Goto-Kakizaki (GK) rats were used as diabetic models. Isolated mesenteric arteries (MAs) and vascular smooth muscle cells (VSMCs) from rat models were used to assess K+-induced arterial constriction and CaV1.2 channel functions using vascular myograph and whole-cell patch clamp, respectively. K+-induced vasoconstriction is persistently enhanced in the MAs from diabetic rats, and CaV1.2 alternative spliced exon 9* is increased, while exon 33 is decreased in rat diabetic arteries. Furthermore, CaV1.2 channels exhibit hyperpolarized current–voltage and activation curve in VSMCs from diabetic rats, which facilitates the channel function. Unexpectedly, the application of glycated serum (GS), mimicking advanced glycation end-products (AGEs), but not glucose, downregulates the expression of the splicing factor Rbfox1 in VSMCs. Moreover, GS application or Rbfox1 knockdown dynamically regulates alternative exons 9* and 33, leading to facilitated functions of CaV1.2 channels in VSMCs and MAs. Notably, GS increases K+-induced intracellular calcium concentration of VSMCs and the vasoconstriction of MAs. These results reveal that AGEs, not glucose, long-termly regulates CaV1.2 alternative splicing events by decreasing Rbfox1 expression, thereby enhancing channel functions and increasing vasoconstriction under diabetic hyperglycemia. This study identifies the specific molecular mechanism for enhanced vasoconstriction under hyperglycemia, providing a potential target for managing diabetic vascular complications. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-024-05198-z.


Figure S1 .
Figure S1.Overexpression of Rbfox1 in mesenteric arteries reduces vasoconstriction in Goto-Kakizaki (GK) rats.(A) Random and fasting blood glucose levels in the rats.(B) Expression of Rbfox1 was detected 3 days after treatment by Rbfox1 expression plasmids (OE) in mesenteric arteries from GK rats.β-actin served as an internal control.Band intensities were analyzed to show the relative expression of Rbfox1 and summarized as a bar chart.n=6 rats for each group, 1-way ANOVA followed by a Tukey's post hoc test.(C) Exemplary traces of mesenteric artery tension responding to increased KCl extracellular concentration (from 0 to 60 mmol/L KCl) using vascular myograph in Wistar, GK and Rbfox1 overexpressed GK rats.(D) Plots of concentration-tension relationship were represented and fitted with Boltzmann equation for differently-treated arteries.n=6 rats for each group.*P<0.05vs Wistar rats, 2-way ANOVA followed by Sidak's multiple comparisons.

Figure S2 .
Figure S2.MAs from diabetic rats are more sensitive to nifedipine in comparison to control ones.(A) Arterial tension responding to different concentration of nifedipine under 60 mmol/L KCl bath solution was measured by vascular myography in control (Ctrl) or HFD/STZ-treated rats (one artery for each rat).(B) Plots of concentration-tension relationship were represented for the MAs from control or diabetic rats.**P<0.01vs control rats, 2-way ANOVA followed by Sidak's multiple comparisons.

Figure S3 .
Figure S3.The cell capacitances (C m ) are measured in differentially-treated VSMCs.The Cm were shown as scatter plots in freshly isolated VSMCs of MAs from control or HFD/STZ-treated rats (A), in VSMCs treated with NG, GS or GS plus H-89 (B), in VSMCs treated with vehicle (Ctrl), MGO or MGO plus H-89 (C), and in NT or Rbfox1 siRNA-treated VSMCs (D).*P<0.05,ns indicates no significant differences, unpaired t test or 1-way ANOVA followed by a Tukey's post hoc test.

Figure S4 .
Figure S4.Ca V 1.2 channel is aberrantly spliced in the arteries from GK rats and nonhypertensive HFD/STZ rats.Mesenteric arteries (MAs) from Wistar or GK rats, and Ctrl or HFD/STZ rats were isolated to determine the expression level of Rbfox1 protein by Western blotting.The proportion of CaV1.2 with exon 8/8a, exon 9* or exon 33 were detected by RT-PCR.The relative Rbfox1 expression was normalized to β-actin after treatments (A, F).The values for percent exon 8/8a (B, G), exon 9* (C, H) or exon 33 inclusion (D, I) of CaV1.2 channels were presented as bar charts.Relative expression of Rbfox1 and summarized as a bar chart (E, J). n=3-6 rats for each group.ns indicates no significant differences, *P<0.05,**P<0.01vs Wistar or Ctrl rats, unpaired t test.

Figure S5 .
Figure S5.Rbfox1 is downregulated in the arteries/VSMCs from HFD/STZ rats.Immunofluorescence staining was used to detect the expression of Rbfox1 in MAs (A) or isolated VSMCs (B) from Ctrl and HFD/STZ rats.Fluorescence intensities were analyzed (C&D).(E) Fluorescence intensity profiles for Rbfox1 (red) and DAPI (blue) along the yellow line drawn in the pictures, expressed as Arbitrary Unit (AU), in Ctrl and HFD/STZ rats.(n=3 arteries and 12 cells from 3 rats, respectively).*P<0.05,**P<0.01vs ctrl, unpaired t test.

Figure S6 .
Figure S6.Rbfox1 is upregulated in the arteries from diabetic patients.(A) Blood glucose levels from the patients with or without diabetes.**P=0.0048vs non-diabetic patients, unpaired t test.(B) Rbfox1 protein expression detected by Western blotting in diabetic versus nondiabetic cerebral arteries from the patients, β-actin was served as an internal control.(C) The relative Rbfox1 expression was normalized to β-actin, and presented as a bar chart.*P=0.022vs non-diabetic patients, unpaired t test with Welch's correction.

Figure S7 .
Figure S7.GS decreases Rbfox1 expression and regulates Ca V 1.2 alternative exons 9* and 33.(A) Mannitol (25 mmol/L) was used to treat isolated VSMCs for 48 hr.The expression of Rbfox1 was checked by Western blotting, and β-actin was detected as internal control.(B) The expression level of Rbfox1 was normalized to β-actin expression after mannitol treatment.P=0.7304, unpaired t test.(C) Rbfox1 expression was checked by Western blotting after treating with increasing concentration of GS. (D) The relative Rbfox1 expression levels were normalized by β-actin expression.(E) Expressions of CaV1.2 alternative exon 9* or exon 33 were checked by RT-PCR.The values for percent exon 9* (F) or exon 33 inclusion (G) were analyzed after treating with GS. *P<0.05,**P<0.01vs NG-treated VSMCs, 1-way ANOVA followed by a Tukey's post hoc test.

Figure S8 .
Figure S8.Cell culture does not alter Ca V 1.2 AS events in isolated VSMCs.(A) Freshly isolated VSMCs from rats (0 hr) were cultured with the medium for 24 or 48 hr.The proportion of CaV1.2 with alternative exon 9* (B) or exon 33 (C) were detected by RT-PCR.Gapdh mRNA was detected as loading control.ns indicates no significant differences, 1-way ANOVA followed by a Tukey's post hoc test.(D) Freshly isolated VSMCs from control or HFD/STZ rats (0 hr) were cultured with the medium for 24 or 48 hr.The proportion of CaV1.2 with alternative exon 9* (B) or exon 33 (C) were detected by RT-PCR.Gapdh mRNA was detected as loading control.**P<0.01vs control rats, 2-way ANOVA followed by a Sidak's multiple comparisons.

Figure S9 .
Figure S9.GS application does not increase Ca V 1.2 expression and S1928 phosphorylation in VSMCs.(A) VSMCs were treated with 20% NG, GS or GS plus 50 μmol/L PKA inhibitor H-89 for 48 hr and Western blotting were performed to detect the expression of total CaV1.2 (B) and S1928 phosphorylated CaV1.2 (C).**P<0.01vs GS. ns indicates no significant differences, 1-way ANOVA followed by a Tukey's post hoc test.

Figure S10 .
Figure S10.Methylglyoxal (MGO) induces decreased Rbfox1, aberrant Ca V 1.2 AS and hyperpolarization of current-voltage curve of Ca V 1.2 in VSMCs.(A) As one of main components in AGEs, MGO (500 μmol/L) was used to treat VSMCs for 48 hr and β-actin was detected as internal control.The proportion of CaV1.2 with alternative exon 9* or exon 33 were detected by RT-PCR.(B) The relative Rbfox1 expression level was presented in differently-treated VSMCs.The proportions of CaV1.2 with alternative exon 9* (C) or exon 33 (D) were analyzed in differently treated VSMCs.(E) Whole-cell currents in the CaV1.2 channel were recorded under different potentials, increasing from −50 to 50 mV (10-mV increase per step; I-V protocol) in isolated aortic VSMCs under 10 mmol/L Ba 2+ bath solution.Additionally, 50 μmol/L PKA inhibitor H-89 was applied 2 hr before the CaV1.2 currents recording in MGO-treated VSMCs.(F) Plots of CaV1.2 current-voltage relationship (I-V) curve of VSMCs were analyzed after treating with vehicle (Ctrl), MGO or MGO plus H-89.(G) Current densities of CaV1.2 channel were calculated by currents divided by cell capacitance (Cm) of the VSMCs.

Figure S11 .
Figure S11.Rbfox1 dynamically regulates Ca V 1.2 alternative exons 9* and 33 in MAs.(A) NT or Rbfox1 siRNAs were transfected into isolated MAs using reversible permeabilization procedure.After 72 hr culture, Rbfox1 protein and Cav1.2 with alternative exon 9* or exon 33 were detected by Western blotting and RT-PCR, respectively.(B) The relative Rbfox1 expression was presented in MAs after different treatments.The values for percent exon 9* (C) or exon 33 inclusion (D) were presented as bar charts.*P<0.05,**P<0.01vs NT siRNA-treated MAs, unpaired t test.

Figure
Figure S12.H-89 does not affect Rbfox1 expression and Ca V 1.2 AS events in NG-treated VSMCs.(A)Vehicle (Ctrl) or H-89 (50 μmol/L) was used to treat isolated VSMCs for 48 hr.The expression of Rbfox1 was checked by Western blotting, and the proportion of CaV1.2 with alternative exon 9* or exon 33 were detected by RT-PCR.(B) The relative Rbfox1 expression was normalized to β-actin after treatments.The values for percent exon 9* (C) or exon 33 inclusion (D) of CaV1.2 channels were presented as bar charts.ns indicates no significant differences, unpaired t test.

Figure S14 .
Figure S14.PKA inhibitor H-89 doesn't affect GS-induced vasoconstriction.(A) Isolated MAswere cultured with 20% GS with or without H-89 (50 μmol/L) for 48 hr, then the forced tension induced by increasing extracellular K + concentration was recorded by using vascular myograph.(B) Plots of concentration-tension relationship were represented and fitted with Boltzmann equation for differently-treated arteries.There were no significant statistical differences, 2-way ANOVA followed by Sidak's multiple comparisons.